Compounds and compositions as protein kinase inhibitors

ABSTRACT

The invention provides a novel class of compounds, pharmaceutical compositions comprising such compounds and methods of using such compounds to treat or prevent diseases or disorders associated with abnormal or deregulated kinase activity, particularly diseases or disorders that involve abnormal activation of the Abl, BCR-Abl, PDGF-R, lck, SAPK2α, p38, TGFβ, KDR, c-Kit, b-RAF, c-RAF, FLT1 and FLT4 kinases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is U.S. Divisional Patent Application of U.S. patentapplication Ser. No. 10/956,880, filed Oct. 1, 2004 which claims thebenefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional PatentApplication No. 60/508,450, filed Oct. 2, 2003. The disclosures of whichare incorporated herein by reference in their entirety and for allpurposes.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention provides a novel class of compounds, pharmaceuticalcompositions comprising such compounds and methods of using suchcompounds to treat or prevent diseases or disorders associated withabnormal or deregulated kinase activity, particularly diseases ordisorders that involve abnormal activation of the Abl, BCR-Abl, lck,SAPK2α, PDGF-R, p38, TGFβ, KDR, c-Kit, b-RAF, c-RAF, FLT1 and FLT4kinases.

2. Background

The protein kinases represent a large family of proteins, which play acentral role in the regulation of a wide variety of cellular processesand maintaining control over cellular function. A partial, non-limiting,list of these kinases include: receptor tyrosine kinases such asplatelet-derived growth factor receptor kinase (PDGF-R), TGFβ,VEGF-receptor kinase (e.g. KDR, Flt-1 and Flt-4), the receptor kinasefor stem cell factor, c-kit; non-receptor tyrosine kinases such Abl andthe fusion kinase BCR-Abl; and serine/threonine kinases such as p38,b-RAF and c-RAF. Aberrant kinase activity has been observed in manydisease states including benign and malignant proliferative disorders aswell as diseases resulting from inappropriate activation of the immuneand nervous systems.

The novel compounds of this invention inhibit the activity of one ormore protein kinases and are, therefore, expected to be useful in thetreatment of kinase-associated diseases.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides compounds of Formula I:

in which:

R₁ and R₂ are independently selected from hydrogen, —XR₃, —XC(O)R₃,—XSR₃, —XS(O)R₃, —XS(O)₂R₃, —XOR₄, —XNC(O)NHR₃R₄ and —XOC(O)NR₃R₄;wherein X is a bond or C₁₋₄alkylene; R₄ is selected from hydrogen andC₁₋₆alkyl; R₃ is selected from C₆₋₁₀aryl, C₅₋₁₀heteroaryl,C₃₋₁₂cycloalkyl and C₃₋₈heterocycloalkyl; wherein any aryl, heteroaryl,cycloalkyl or heterocycloalkyl of R₃ is optionally substituted by 1 to 3radicals selected from hydroxy, halo, nitro, C₁₋₆alkyl, C₁₋₆alkoxy,halo-substituted-C₁₋₆alkyl, halo-substituted-C₁₋₆alkoxy, —XC(O)OR₄ and—NR₄R₅; wherein X is a bond or C₁₋₄alkylene and R₄ and R₅ areindependently selected from hydrogen and C₁₋₆alkyl; wherein thepyridinyl ring A can have up to three —C═ groups replaced with —N═groups; wherein the naphthyl ring B can have up to four —C═ groupsreplaced with —N═ groups; and the N-oxide derivatives, prodrugderivatives, protected derivatives, individual isomers and mixture ofisomers thereof; and the pharmaceutically acceptable salts and solvates(e.g. hydrates) of such compounds.

In a second aspect, the present invention provides a pharmaceuticalcomposition which contains a compound of Formula I or a N-oxidederivative, individual isomers and mixture of isomers thereof; or apharmaceutically acceptable salt thereof, in admixture with one or moresuitable excipients.

In a third aspect, the present invention provides a method of treating adisease in an animal in which inhibition of kinase activity,particularly Abl, BCR-Abl, PDGF-R, lck, SAPK2α, p38, TGFβ, KDR, c-Kit,b-RAF, c-RAF, FLT1 and FLT4 activity, can prevent, inhibit or amelioratethe pathology and/or symptomology of the diseases, which methodcomprises administering to the animal a therapeutically effective amountof a compound of Formula I or a N-oxide derivative, individual isomersand mixture of isomers thereof, or a pharmaceutically acceptable saltthereof.

In a fourth aspect, the present invention provides the use of a compoundof Formula I in the manufacture of a medicament for treating a diseasein an animal in which kinase activity, particularly Abl, BCR-Abl,PDGF-R, lck, SAPK2α, p38, TGFβ, KDR, c-Kit, b-RAF, c-RAF, FLT1 and FLT4activity, contributes to the pathology and/or symptomology of thedisease.

In a fifth aspect, the present invention provides a process forpreparing compounds of Formula I and the N-oxide derivatives, prodrugderivatives, protected derivatives, individual isomers and mixture ofisomers thereof, and the pharmaceutically acceptable salts thereof.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Alkyl” as a group and as a structural element of other groups, forexample halo-substituted-alkyl and alkoxy, can be eitherstraight-chained or branched. C₁₋₄-alkoxy includes, methoxy, ethoxy, andthe like. Halo-substituted alkyl includes trifluoromethyl,pentafluoroethyl, and the like.

“Aryl” means a monocyclic or fused bicyclic aromatic ring assemblycontaining six to ten ring carbon atoms. For example, aryl may be phenylor naphthyl, preferably phenyl. “Arylene” means a divalent radicalderived from an aryl group. “Heteroaryl” is as defined for aryl whereone or more of the ring members are a heteroatom. For example heteroarylincludes pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl,benzofuranyl, benzopyranyl, benzothiopyranyl, benzo[1,3]dioxole,imidazolyl, benzo-imidazolyl, pyrimidinyl, furanyl, oxazolyl,isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, thienyl, etc.

“Cycloalkyl” means a saturated or partially unsaturated, monocyclic,fused bicyclic or bridged polycyclic ring assembly containing the numberof ring atoms indicated. For example, C₃₋₁₀cycloalkyl includescyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.“Heterocycloalkyl” means cycloalkyl, as defined in this application,provided that one or more of the ring carbons indicated, are replaced bya moiety selected from —O—, —N═, —NR—, —C(O)—, —S—, —S(O)— or —S(O)₂—,wherein R is hydrogen, C₁₋₄alkyl or a nitrogen protecting group. Forexample, C₃₋₈heterocycloalkyl as used in this application to describecompounds of the invention includes morpholino, pyrrolidinyl,piperazinyl, piperidinyl, piperidinylone,1,4-dioxa-8-aza-spiro[4.5]dec-8-yl, etc.

“Halogen” (or halo) preferably represents chloro or fluoro, but may alsobe bromo or iodo.

“Treat”, “treating” and “treatment” refer to a method of alleviating orabating a disease and/or its attendant symptoms.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides compounds, compositions and methods forthe treatment of kinase related disease, particularly Abl, BCR-Abl,PDGF-R, lck, SAPK2α, p38, TGFβ, KDR, c-Kit, b-RAF, c-RAF, FLT1 and FLT4kinase related diseases. For example, leukemia and other proliferationdisorders related to BCR-Abl can be treated through the inhibition ofwild type and mutant forms of Bcr-Abl.

In one embodiment, with reference to compounds of Formula I, R₁ and R₂are independently selected from hydrogen, —XR₃ and —XOC(O)NR₃R₄; whereinX is a bond or C₁₋₄alkylene; R₄ is selected from hydrogen and C₁₋₆alkyl;R₃ is selected from C₆₋₁₀aryl, C₅₋₁₀heteroaryl and C₃₋₈heterocycloalkyl;wherein any aryl, heteroaryl or heterocycloalkyl of R₃ is optionallysubstituted by 1 to 3 radicals selected from halo, nitro, C₁₋₆alkyl,C₁₋₆alkoxy, halo-substituted-C₁₋₆alkyl, —XC(O)OR₄ and —NR₄R₅; wherein Xis a bond or C₁₋₄alkylene and R₄ and R₅ are independently selected fromhydrogen and C₁₋₆alkyl.

In another embodiment, R₁ is selected from hydrogen and phenyl; whereinsaid phenyl is optionally substituted by 1 to 3 radicals selected fromtrifluoromethyl, dimethylamino, methoxy, halo, ethoxy and nitro.

In a further embodiment, R₂ is selected from hydrogen, —OH and—OC(O)NHR₄; wherein R₄ is selected from phenyl andbenzo[1,3]dioxol-5-yl; wherein said phenyl of R₄ is optionallysubstituted by 1 to 3 radicals selected from —C(O)OCH₃ anddimethylamino.

Preferred compounds of Formula I are selected from: Phenyl-carbamic acid8-(2-phenyl-5-pyridin-4-yl-3H-imidazol-4-yl)-naphthalen-2-yl ester;4-(5-Naphthalen-1-yl-2-phenyl-3H-imidazol-4-yl)-pyridine;4-[2-(3,5-Bis-trifluoromethyl-phenyl)-5-naphthalen-1-yl-3H-imidazol-4-yl]-pyridine;Dimethyl-[4-(4-naphthalen-1-yl-5-pyridin-4-yl-1H-imidazol-2-yl)-phenyl]-amine;4-[5-Naphthalen-1-yl-2-(2,4,6-trimethoxy-phenyl)-3H-imidazol-4-yl]-pyridine;4-[2-(2-Fluoro-phenyl)-5-naphthalen-1-yl-3H-imidazol-4-yl]-pyridine;4-[2-(2-Ethoxy-phenyl)-5-naphthalen-1-yl-3H-imidazol-4-yl]-pyridine;4-[5-Naphthalen-1-yl-2-(3-nitro-phenyl)-3H-imidazol-4-yl]-pyridine;4-(5-Naphthalen-1-yl-3H-imidazol-4-yl)-pyridine; Phenyl-carbamic acid6-(2-phenyl-5-pyridin-4-yl-1H-imidazol-4-yl)-naphthalen-2-yl ester;6-(2-Phenyl-5-pyridin-4-yl-1H-imidazol-4-yl)-naphthalen-2-ol;4-[6-(2-Phenyl-5-pyridin-4-yl-1H-imidazol-4-yl)-naphthalen-2-yloxycarbonylamino]-benzoicacid methyl ester; Benzo[1,3]dioxol-5-yl-carbamic acid6-(2-phenyl-5-pyridin-4-yl-1H-imidazol-4-yl)-naphthalen-2-yl ester;5-[6-(2-Phenyl-5-pyridin-4-yl-1H-imidazol-4-yl)-naphthalen-2-yloxycarbonylamino]-isophthalicacid dimethyl ester; (4-Dimethylamino-phenyl)-carbamic acid6-(2-phenyl-5-pyridin-4-yl-1H-imidazol-4-yl)-naphthalen-2-yl ester;Benzo[1,3]dioxol-5-yl-carbamic acid8-(2-phenyl-5-pyridin-4-yl-1H-imidazol-4-yl)-naphthalen-2-yl ester;5-[8-(2-Phenyl-5-pyridin-4-yl-1H-imidazol-4-yl)-naphthalen-2-yloxycarbonylamino]-isophthalicacid dimethyl ester; and (4-Dimethylamino-phenyl)-carbamic acid8-(2-phenyl-5-pyridin-4-yl-1H-imidazol-4-yl)-naphthalen-2-yl ester.

Further preferred compounds of Formula I are detailed in the Examplesand Table I, infra.

Pharmacology and Utility

Compounds of the invention modulate the activity of protein tyrosinekinases and, as such, are useful for treating diseases or disorders inwhich protein tyrosine kinases, particularly Abl, BCR-Abl, PDGF-R, lck,SAPK2α, p38, TGFβ, KDR, c-Kit, b-RAF, c-RAF, FLT1 and FLT4 kinases,contribute to the pathology and/or symptomology of the disease.

Abelson tyrosine kinase (i.e. Abl, c-Abl) is involved in the regulationof the cell cycle, in the cellular response to genotoxic stress, and inthe transmission of information about the cellular environment throughintegrin signaling. Overall, it appears that the Abl protein serves acomplex role as a cellular module that integrates signals from variousextracellular and intracellular sources and that influences decisions inregard to cell cycle and apoptosis. Abelson tyrosine kinase includessub-types derivatives such as the chimeric fusion (oncoprotein) BCR-Ablwith deregulated tyrosine kinase activity or the v-Abl. BCR-Abl iscritical in the pathogenesis of 95% of chronic myelogenous leukemia(CML) and 10% of acute lymphocytic leukemia. STI-571 (Gleevec) is aninhibitor of the oncogenic BCR-Abl tyrosine kinase and is used for thetreatment of chronic myeloid leukemia (CML). However, some patients inthe blast crisis stage of CML are resistant to STI-571 due to mutationsin the BCR-Abl kinase. Over 22 mutations have been reported to date withthe most common being G250E, E255V, T3151, F317L and M351T.

Compounds of the present invention inhibit abl kinase, especially v-ablkinase. The compounds of the present invention also inhibit wild-typeBCR-Abl kinase and mutations of BCR-Abl kinase and are thus suitable forthe treatment of Bcr-abl-positive cancer and tumor diseases, such asleukemias (especially chronic myeloid leukemia and acute lymphoblasticleukemia, where especially apoptotic mechanisms of action are found),and also shows effects on the subgroup of leukemic stem cells as well aspotential for the purification of these cells in vitro after removal ofsaid cells (for example, bone marrow removal) and reimplantation of thecells once they have been cleared of cancer cells (for example,reimplantation of purified bone marrow cells).

PDGF (Platelet-derived Growth Factor) is a very commonly occurringgrowth factor, which plays an important role both in normal growth andalso in pathological cell proliferation, such as is seen incarcinogenesis and in diseases of the smooth-muscle cells of bloodvessels, for example in atherosclerosis and thrombosis. Compounds of theinvention can inhibit PDGF receptor (PDGFR) activity and are, therefore,suitable for the treatment of tumor diseases, such as gliomas, sarcomas,prostate tumors, and tumors of the colon, breast, and ovary.

Compounds of the present invention, can be used not only as atumor-inhibiting substance, for example in small cell lung cancer, butalso as an agent to treat non-malignant proliferative disorders, such asatherosclerosis, thrombosis, psoriasis, scleroderma and fibrosis, aswell as for the protection of stem cells, for example to combat thehemotoxic effect of chemotherapeutic agents, such as 5-fluoruracil, andin asthma. Compounds of the invention can especially be used for thetreatment of diseases, which respond to an inhibition of the PDGFreceptor kinase.

Compounds of the present invention show useful effects in the treatmentof disorders arising as a result of transplantation, for example,allogenic transplantation, especially tissue rejection, such asespecially obliterative bronchiolitis (OB), i.e. a chronic rejection ofallogenic lung transplants. In contrast to patients without OB, thosewith OB often show an elevated PDGF concentration in bronchioalveolarlavage fluids.

Compounds of the present invention are also effective in diseasesassociated with vascular smooth-muscle cell migration and proliferation(where PDGF and PDGF-R often also play a role), such as restenosis andatherosclerosis. These effects and the consequences thereof for theproliferation or migration of vascular smooth-muscle cells in vitro andin vivo can be demonstrated by administration of the compounds of thepresent invention, and also by investigating its effect on thethickening of the vascular intima following mechanical injury in vivo.

The compounds of the present invention also inhibit cellular processesinvolving stem-cell factor (SCF, also known as the c-kit ligand or steelfactor), such as inhibiting SCF receptor (kit) autophosphorylation andSCF-stimulated activation of MAPK kinase (mitogen-activated proteinkinase). MO7e cells are a human promegakaryocytic leukemia cell line,which depends on SCF for proliferation. Compounds of the invention caninhibit the autophosphorylation of SCF receptors.

The Ras-Raf-MEK-ERK signaling pathway mediates cellular response togrowth signals. Ras is mutated to an oncogenic form in ˜15% of humancancer. The Raf family belongs to the serine/threonine protein kinaseand it includes three members, A-Raf, B-Raf and c-Raf (or Raf-1). Thefocus on Raf being a drug target has centered on the relationship of Rafas a downstream effector of Ras. However, recent data suggests thatB-Raf may have a prominent role in the formation of certain tumors withno requirement for an activated Ras allele (Nature 417, 949-954 (1 Jul.2002). In particular, B-Raf mutations have been detected in a largepercentage of malignant melanomas.

Existing medical treatments for melanoma are limited in theireffectiveness, especially for late stage melanomas. The compounds of thepresent invention also inhibit cellular processes involving b-Rafkinase, providing a new therapeutic opportunity for treatment of humancancers, especially for melanoma.

The compounds of the present invention also inhibit cellular processesinvolving c-Raf kinase. c-Raf is activated by the ras oncogene, which ismutated in a wide number of human cancers. Therefore inhibition of thekinase activity of c-Raf may provide a way to prevent ras mediated tumorgrowth [Campbell, S. L., Oncogene, 17, 1395 (1998)].

The compounds of the present invention also inhibit cellular processesinvolving KDR, Flt-1 and Flt-4. A number of diseases are known which areassociated with deregulated angiogenesis, for example diseases caused byocular neovascularisation, especially retinopathies (diabeticretinopathy, age-related macular degeneration); psoriasis;haemangioblastomas, such as “strawberry-marks” (=haemangioma); variousinflammatory diseases, such as arthritis, especially rheumatoidarthritis, arterial atherosclerosis and atherosclerosis occurring aftertransplants, endometriosis or chronic asthma; and, especially, tumordiseases (solid tumors, but also leukemias and other liquid tumors,since many primitive blood cells and leukemia cells express c-kit, KDR,Flt-1 and Flt-4). Flt-4 is expressed in developing lymphatic vessels.Only the lymphatic endothelia and some high endothelial venules expressFlt4 mRNA in adult human tissues and increased expression occurs inlymphatic sinuses in metastatic lymph nodes and in lymphangioma.Inhibition of KDR-mediated functional effects by inhibiting KDR'scatalytic activity is considered to be an important therapeutic strategyin the treatment of angiogenized disease states including cancer.

Multiple forms of p38 MAPK (α, β, γ, δ), each encoded by a separategene, form part of a kinase cascade involved in the response of cells toa variety of stimuli, including osmotic stress, UV light and cytokinemediated events. These four isoforms of p38 are thought to regulatedifferent aspects of intracellular signaling. Its activation is part ofa cascade of signaling events that lead to the synthesis and productionof pro-inflammatory cytokines like TNFα. P38 functions byphosphorylating downstream substrates that include other kinases andtranscription factors. Agents that inhibit p38 kinase have been shown toblock the production of cytokines including but not limited to TNFα,IL-6, IL-8 and IL-1β. Peripheral blood monocytes (PBMCs) have been shownto express and secrete pro-inflammatory cytokines when stimulated withlipopolysaccharide (LPS) in vitro. P38 inhibitors efficiently block thiseffect when PBMCs are pretreated with such compounds prior tostimulation with LPS. P38 inhibitors are efficacious in animal models ofinflammatory disease. The destructive effects of many disease states arecaused by the over production of pro-inflammatory cytokines. The abilityof p38 inhibitors to regulate this overproduction makes them useful asdisease modifying agents.

Molecules that block p38's function have been shown to be effective ininhibiting bone resorption, inflammation, and other immune andinflammation-based pathologies. Thus, a safe and effective p38 inhibitorwould provide a means to treat debilitating diseases that can beregulated by modulation of p38 signaling like, for example, RA.Therefore, compounds of the invention that inhibit p38 activity areuseful for the treatment of inflammation, osteoarthritis, rheumatoidarthritis, cancer, autoimmune diseases, and for the treatment of othercytokine mediated diseases.

Transforming growth factor-beta (TGFβ) denotes a superfamily of proteinsthat includes, for example, TGFβ1, TGFβ2, and TGFβ3, which arepleotropic modulators of cell growth and differentiation, embryonic andbone development, extracellular matrix formation, hematopoiesis, immuneand inflammatory responses. The members of the TGFβ family initiateintracellular signaling pathways leading ultimately to the expression ofgenes that regulate the cell cycle, control proliferative responses, orrelate to extracellular matrix proteins that mediate outside-in cellsignaling, cell adhesion, migration and intercellular communication.Consequently, compounds of the invention that are inhibitors of the TGFβintracellular signaling pathway are useful treatments forfibroproliferative diseases, including kidney disorders associated withunregulated TGFβ activity and excessive fibrosis includingglomerulonephritis (GN), such as mesangial proliferative GN, immune GN,and crescentic GN. Other renal conditions include diabetic nephropathy,renal interstitial fibrosis, renal fibrosis in transplant patientsreceiving cyclosporin, and HIV-associated nephropathy. Collagen vasculardisorders include progressive systemic sclerosis, polymyositis,scleroderma, dermatomyositis, eosinophilic fascitis, morphea, or thoseassociated with the occurrence of Raynaud's syndrome. Lung fibrosesresulting from excessive TGFβ activity include adult respiratorydistress syndrome, COPD, idiopathic pulmonary fibrosis, and interstitialpulmonary fibrosis often associated with autoimmune disorders, such assystemic lupus erythematosus and scleroderma, chemical contact, orallergies. Another autoimmune disorder associated withfibroproliferative characteristics is rheumatoid arthritis.Fibroproliferative conditions can be associated with surgical eyeprocedures. Such procedures include retinal reattachment surgeryaccompanying proliferative vitreoretinopathy, cataract extraction withintraocular lens implantation, and post glaucoma drainage surgery.

In accordance with the foregoing, the present invention further providesa method for preventing or treating any of the diseases or disordersdescribed above in a subject in need of such treatment, which methodcomprises administering to said subject a therapeutically effectiveamount (See, “Administration and Pharmaceutical Compositions”, infra) ofa compound of Formula I or a pharmaceutically acceptable salt thereof.For any of the above uses, the required dosage will vary depending onthe mode of administration, the particular condition to be treated andthe effect desired.

Administration and Pharmaceutical Compositions

In general, compounds of the invention will be administered intherapeutically effective amounts via any of the usual and acceptablemodes known in the art, either singly or in combination with one or moretherapeutic agents. A therapeutically effective amount may vary widelydepending on the severity of the disease, the age and relative health ofthe subject, the potency of the compound used and other factors. Ingeneral, satisfactory results are indicated to be obtained systemicallyat daily dosages of from about 0.03 to 2.5 mg/kg per body weight. Anindicated daily dosage in the larger mammal, e.g. humans, is in therange from about 0.5 mg to about 100 mg, conveniently administered, e.g.in divided doses up to four times a day or in retard form. Suitable unitdosage forms for oral administration comprise from ca. 1 to 50 mg activeingredient.

Compounds of the invention can be administered as pharmaceuticalcompositions by any conventional route, in particular enterally, e.g.,orally, e.g., in the form of tablets or capsules, or parenterally, e.g.,in the form of injectable solutions or suspensions, topically, e.g., inthe form of lotions, gels, ointments or creams, or in a nasal orsuppository form. Pharmaceutical compositions comprising a compound ofthe present invention in free form or in a pharmaceutically acceptablesalt form in association with at least one pharmaceutically acceptablecarrier or diluent can be manufactured in a conventional manner bymixing, granulating or coating methods. For example, oral compositionscan be tablets or gelatin capsules comprising the active ingredienttogether with a) diluents, e.g., lactose, dextrose, sucrose, mannitol,sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum,stearic acid, its magnesium or calcium salt and/or polyethyleneglycol;for tablets also c) binders, e.g., magnesium aluminum silicate, starchpaste, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose and or polyvinylpyrrolidone; if desired d)disintegrants, e.g., starches, agar, alginic acid or its sodium salt, oreffervescent mixtures; and/or e) absorbents, colorants, flavors andsweeteners. Injectable compositions can be aqueous isotonic solutions orsuspensions, and suppositories can be prepared from fatty emulsions orsuspensions. The compositions may be sterilized and/or containadjuvants, such as preserving, stabilizing, wetting or emulsifyingagents, solution promoters, salts for regulating the osmotic pressureand/or buffers. In addition, they may also contain other therapeuticallyvaluable substances. Suitable formulations for transdermal applicationsinclude an effective amount of a compound of the present invention witha carrier. A carrier can include absorbable pharmacologically acceptablesolvents to assist passage through the skin of the host. For example,transdermal devices are in the form of a bandage comprising a backingmember, a reservoir containing the compound optionally with carriers,optionally a rate controlling barrier to deliver the compound to theskin of the host at a controlled and predetermined rate over a prolongedperiod of time, and means to secure the device to the skin. Matrixtransdermal formulations may also be used. Suitable formulations fortopical application, e.g., to the skin and eyes, are preferably aqueoussolutions, ointments, creams or gels well-known in the art. Such maycontain solubilizers, stabilizers, tonicity enhancing agents, buffersand preservatives.

Compounds of the invention can be administered in therapeuticallyeffective amounts in combination with one or more therapeutic agents(pharmaceutical combinations). For example, synergistic effects canoccur with other immunomodulatory or anti-inflammatory substances, forexample when used in combination with cyclosporin, rapamycin, orascomycin, or immunosuppressant analogues thereof, for examplecyclosporin A (CsA), cyclosporin G, FK-506, rapamycin, or comparablecompounds, corticosteroids, cyclophosphamide, azathioprine,methotrexate, brequinar, leflunomide, mizoribine, mycophenolic acid,mycophenolate mofetil, 15-deoxyspergualin, immunosuppressant antibodies,especially monoclonal antibodies for leukocyte receptors, for exampleMHC, CD2, CD3, CD4, CD7, CD25, CD28, B7, CD45, CD58 or their ligands, orother immunomodulatory compounds, such as CTLA41g. Where the compoundsof the invention are administered in conjunction with other therapies,dosages of the co-administered compounds will of course vary dependingon the type of co-drug employed, on the specific drug employed, on thecondition being treated and so forth.

The invention also provides for a pharmaceutical combinations, e.g. akit, comprising a) a first agent which is a compound of the invention asdisclosed herein, in free form or in pharmaceutically acceptable saltform, and b) at least one co-agent. The kit can comprise instructionsfor its administration.

The terms “co-administration” or “combined administration” or the likeas utilized herein are meant to encompass administration of the selectedtherapeutic agents to a single patient, and are intended to includetreatment regimens in which the agents are not necessarily administeredby the same route of administration or at the same time.

The term “pharmaceutical combination” as used herein means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that the activeingredients, e.g. a compound of Formula I and a co-agent, are bothadministered to a patient simultaneously in the form of a single entityor dosage. The term “non-fixed combination” means that the activeingredients, e.g. a compound of Formula I and a co-agent, are bothadministered to a patient as separate entities either simultaneously,concurrently or sequentially with no specific time limits, wherein suchadministration provides therapeutically effective levels of the 2compounds in the body of the patient. The latter also applies tococktail therapy, e.g. the administration of 3 or more activeingredients.

Processes for Making Compounds of the Invention

The present invention also includes processes for the preparation ofcompounds of the invention. In the reactions described, it can benecessary to protect reactive functional groups, for example hydroxy,amino, imino, thio or carboxy groups, where these are desired in thefinal product, to avoid their unwanted participation in the reactions.Conventional protecting groups can be used in accordance with standardpractice, for example, see T. W. Greene and P. G. M. Wuts in “ProtectiveGroups in Organic Chemistry”, John Wiley and Sons, 1991.

Compounds of Formula I can be prepared by proceeding as in the followingReaction Scheme I:

in which A, B, R₁ and R₂ are as defined for Formula I in the Summary ofthe Invention.

A compound of the invention (Formula I) can be prepared by reacting acompound of formula 2 with a compound of formula 3 in the presence of asuitable reactant (e.g., ammonium acetate, and the like) and a suitablesolvent (e.g., acetic acid, or the like). The reaction is carried out inthe temperature range of 100 to 140° C. and can take up to 24 hours tocomplete. A detailed description of the synthesis of a compound ofFormula I is set forth in the examples, infra.

Additional Processes for Making Compounds of the Invention

A compound of the invention can be prepared as a pharmaceuticallyacceptable acid addition salt by reacting the free base form of thecompound with a pharmaceutically acceptable inorganic or organic acid.Alternatively, a pharmaceutically acceptable base addition salt of acompound of the invention can be prepared by reacting the free acid formof the compound with a pharmaceutically acceptable inorganic or organicbase. Alternatively, the salt forms of the compounds of the inventioncan be prepared using salts of the starting materials or intermediates.

The free acid or free base forms of the compounds of the invention canbe prepared from the corresponding base addition salt or acid additionsalt from, respectively. For example a compound of the invention in anacid addition salt form can be converted to the corresponding free baseby treating with a suitable base (e.g., ammonium hydroxide solution,sodium hydroxide, and the like). A compound of the invention in a baseaddition salt form can be converted to the corresponding free acid bytreating with a suitable acid (e.g., hydrochloric acid, etc.).

Compounds of the invention in unoxidized form can be prepared fromN-oxides of compounds of the invention by treating with a reducing agent(e.g., sulfur, sulfur dioxide, triphenyl phosphine, lithium borohydride,sodium borohydride, phosphorus trichloride, tribromide, or the like) ina suitable inert organic solvent (e.g. acetonitrile, ethanol, aqueousdioxane, or the like) at 0 to 80° C.

Prodrug derivatives of the compounds of the invention can be prepared bymethods known to those of ordinary skill in the art (e.g., for furtherdetails see Saulnier et al., (1994), Bioorganic and Medicinal ChemistryLetters, Vol. 4, p. 1985). For example, appropriate prodrugs can beprepared by reacting a non-derivatized compound of the invention with asuitable carbamoylating agent (e.g., 1,1-acyloxyalkylcarbanochloridate,para-nitrophenyl carbonate, or the like).

Protected derivatives of the compounds of the invention can be made bymeans known to those of ordinary skill in the art. A detaileddescription of techniques applicable to the creation of protectinggroups and their removal can be found in T. W. Greene, “ProtectingGroups in Organic Chemistry”, 3^(rd) edition, John Wiley and Sons, Inc.,1999.

Compounds of the present invention can be conveniently prepared, orformed during the process of the invention, as solvates (e.g.,hydrates). Hydrates of compounds of the present invention can beconveniently prepared by recrystallization from an aqueous/organicsolvent mixture, using organic solvents such as dioxin, tetrahydrofuranor methanol.

Compounds of the invention can be prepared as their individualstereoisomers by reacting a racemic mixture of the compound with anoptically active resolving agent to form a pair of diastereoisomericcompounds, separating the diastereomers and recovering the opticallypure enantiomers. While resolution of enantiomers can be carried outusing covalent diastereomeric derivatives of the compounds of theinvention, dissociable complexes are preferred (e.g., crystallinediastereomeric salts). Diastereomers have distinct physical properties(e.g., melting points, boiling points, solubilities, reactivity, etc.)and can be readily separated by taking advantage of thesedissimilarities. The diastereomers can be separated by chromatography,or preferably, by separation/resolution techniques based upondifferences in solubility. The optically pure enantiomer is thenrecovered, along with the resolving agent, by any practical means thatwould not result in racemization. A more detailed description of thetechniques applicable to the resolution of stereoisomers of compoundsfrom their racemic mixture can be found in Jean Jacques, Andre Collet,Samuel H. Wilen, “Enantiomers, Racemates and Resolutions”, John WileyAnd Sons, Inc., 1981.

In summary, the compounds of Formula I can be made by a process, whichinvolves:

(a) that of reaction schemes I; and

(b) optionally converting a compound of the invention into apharmaceutically acceptable salt;

(c) optionally converting a salt form of a compound of the invention toa non-salt form;

(d) optionally converting an unoxidized form of a compound of theinvention into a pharmaceutically acceptable N-oxide;

(e) optionally converting an N-oxide form of a compound of the inventionto its unoxidized form;

(f) optionally resolving an individual isomer of a compound of theinvention from a mixture of isomers;

(g) optionally converting a non-derivatized compound of the inventioninto a pharmaceutically acceptable prodrug derivative; and

(h) optionally converting a prodrug derivative of a compound of theinvention to its non-derivatized form.

Insofar as the production of the starting materials is not particularlydescribed, the compounds are known or can be prepared analogously tomethods known in the art or as disclosed in the Examples hereinafter.

One of skill in the art will appreciate that the above transformationsare only representative of methods for preparation of the compounds ofthe present invention, and that other well known methods can similarlybe used.

EXAMPLES

The present invention is further exemplified, but not limited, by thefollowing examples that illustrate the preparation of compounds ofFormula I according to the invention.

Example 1 Phenyl-carbamic acid8-(2-phenyl-5-pyridin-4-yl-3H-imidazol-4-yl)-naphthalen-2-yl ester

A mixture of 8-amino-2-naphthol (14.63 g, 91.9 mmol) and di-tert-butyldicarbonate (21.0 g, 96.2 mmol) in methylene chloride (200 mL) andtetrahydrofuran (200 mL) is stirred under reflux condition for 24 hours.The mixture is then cooled to ambient temperature, filtered and thefiltrate is concentrated to dryness. Silica gel chromatography(CH₂Cl₂/EtOEt=5/95 to 10/90) afforded(7-hydroxy-naphthalen-1-yl)-carbamic acid tert-butyl ester (21.50 g, 90%yield); MS m/z (M+Na⁺) 282.05.

To a mixture of (7-hydroxy-naphthalen-1-yl)-carbamic acid tert-butylester (5.11 g, 19.7 mmol) and potassium carbonate (3.26 g, 23.6 mmol) inN,N-dimethylformamide is added benzyl bromide (3 mL, 4.31 g, 25.2 mmol)slowly. The mixture is then stirred at room temperature over night andpoured into ice-water (150 mL). The resulting mixture is extracted withethyl acetate (4×100 mL) and the organic portions are combined, washedwith water and brine, dried over sodium sulfate, concentrated andpurified by silica gel chromatography (Hexanes/EtOAc=15/1) to give(7-benzyloxy-naphthalen-1-yl)-carbamic acid tert-butyl ester (5.93 g,86.3%); ¹H NMR 400 Hz (CDCl₃) δ 1.60 (s, 9H), 5.22 (s, 2H), 6.63 (br,1H), 7.27 (m, 2H), 7.38 (m, 2H), 7.45 (m, 2H), 7.44 (d, J=7.44 Hz, 2H),7.61 (d, J=8.11 Hz, 1H), 7.80 (m, 2H); MS m/z (M+Na⁺) 372.10.

To a solution of (7-benzyloxy-naphthalen-1-yl)-carbamic acid tert-butylester (9.89 g, 28.3 mmol) in methylene chloride (200 mL) is addedtrifluoroacetic acid (50 mL, 73 g, 649 mmol) and the mixture is stirredat room temperature for 30 minutes. The solvent and excessivetrifluoroacetic acid is evaporated under reduced pressure and the crudeproduct is dried on high vacuum over night and the resulting7-benzyloxy-naphthalen-1-ylamine (6.98 g, 99%) was used for next stepwithout further purification.

A solution of 7-benzyloxy-naphthalen-1-ylamine (6.98 g, 28.02 mmol) inconcentrated hydrogen chloride (30 mL), water (30 mL) andtetrahydrofuran (30 mL) is cooled with an ice-salt (sodium chloride)bath. A solution of sodium nitrite (2.32 g, 33.6 mmol) in water (20 mL)is then added in a dropwise manner over a 15-minute period of time andthe reaction temperature is kept under 5° C. The mixture is stirred foranother 30 minutes before potassium iodide (9.3 g, 56.04 mmol) in water(30 mL) is added. The resulting mixture is stirred for three hours atroom temperature before being extracted with ethyl acetate, dried oversodium sulfate, filtered, concentrated and purified by silica gelchromatography (Hexanes/CH₂Cl₂=25/1-20/1) to give7-benzyloxy-1-iodo-naphthalene (6.26 g, 61%); MS m/z (M+Na⁺) 361.00.

To a solution of 7-benzyloxy-1-iodo-naphthalene (4.60 g, 12.77 mmol) inether (120 mL) is added 1.7 M tert-butyl-lithium in pentane (8.26 mL,14.0 mmol) at −78° C. under nitrogen atmosphere and the resultingmixture is stirred for one hour while being warmed up to 5° C. Themixture is cooled to −78° C. again before tetramethylethylene-diamine(1.93 mL, 1.49 g, 12.79 mmol) is added. The mixture is stirred for 10minutes before N,N-dimethylformamide (0.99 mL, 0.93 g, 12.78 mmol) isadded. The mixture is gradually warmed up to room temperature andstirred over night. Ethyl acetate is added to reaction mixture, washedwith saturated ammonium chloride, dried over sodium sulfate, filteredand concentrated. The crude product is purified by silica gelchromatography (Hexanes/EtOAc=20/1) to give7-benzyloxy-naphthalene-1-carbaldehyde (1.95 g, 58.3% yield); ¹H NMR 400Hz (CDCl₃) δ 5.27 (s, 2H), 7.33 (m, 2H), 7.42 (m, 2H), 7.52 (m, 3H),7.83 (d, J=8.98 Hz, 1H), 7.95 (d, J=7.11 Hz, 1H), 8.03 (d, J=8.10 Hz,1H), 10.32 (s, 1H); MS m/z (M+H⁺) 263.05.

To a solution of 4-(tert-butyl-dimethyl-silanyloxymethyl)-pyridine (1.59g, 7.09 mmol) in tetrahydrofuran (40 mL) is added 2M lithiumdiisopropylamide in hexanes (3.55 mL, 7.11 mmol) over 5 minutes at −50°C. under a nitrogen atmosphere. The mixture is stirred at −40° C. forone hour and 7-benzyloxy-naphthalene-1-carbaldehyde (1.69 g, 6.45 mmol)in tetrahydrofuran (20 mL) is added over 10 minutes. The reaction isgradually warmed up to room temperature and stirred at that temperatureover night before being quenched by saturated sodium bicarbonate andextracted with ethyl acetate. The organic layers are combined, washedwith brine, dried over sodium sulfate, filtered and concentrated in avacuum. Silica gel chromatography (Hexanes/EtOAc=2/1-1/1) afforded1-(7-benzyloxy-naphthalen-1-yl)-2-(tert-butyl-dimethyl-silanyloxy)-2-pyridin-4-yl-ethanol(3.85 g, 89% yield); MS m/z (M+H⁺) 486.20.

To a solution of1-(7-benzyloxy-naphthalen-1-yl)-2-(tert-butyl-dimethyl-silanyloxy)-2-pyridin-4-yl-ethanol(3.75 g, 7.72 mmol) in tetrahydrofuran (60 mL) is added 1.0 Mtetrabutylammonium fluoride in tetrahydrofuran (10 mL, 10 mmol) and themixture is stirred at room temperature for 1 hour. The mixture isconcentrated and ethyl acetate is added to the residue. The solution iswashed with water, dried over sodium sulfate, filtered, concentrated anddried on a high vacuum. The resulting diol is used for the followingoxidation without further purification.

A mixture of dimethylsulfoxide (1.64 mL, 1.81 g, 23.1 mmol) andanhydrous methylene chloride (100 mL) is cooled to −70° C. and oxalylchloride (2.0 mL, 2.94 g, 23.1 mmol) is added dropwise. The mixture isstirred for 15 minutes at this temperature and a solution of the abovediol in methylene chloride (40 mL) and dimethylsulfoxide (10 mL) isadded dropwise. The mixture is stirred at −60° C. for 2 hours beforetriethylamine (6.45 mL, 4.68 g, 46.3 mmol) is added. The reactionmixture is gradually warmed up to room temperature and stirred overnight. Water is then added to the mixture and organic phase isseparated, washed with water, dried over sodium sulfate, filtered andconcentrated. The crude product is purified by silica gel chromatography(Hexanes/EtOAC=3/1-2/1) to give1-(7-benzyloxy-naphthalen-1-yl)-2-pyridin-4-yl-ethane-1,2-dione (2.26 g,79.7% yield); ¹H NMR 400 Hz (CDCl₃) δ 5.31 (s, 2H), 7.40 (m, 5H), 7.55(d, J=7.30 Hz, 2H), 7.81 (dd, J=1.66, 4.42 Hz, 2H), 7.87 (m, 2H), 8.10(d, J=8.08 Hz, 1H), 8.89 (dd, J=1.55, 4.48 Hz, 2H), 8.93 (d, J=2.48 Hz,1H); MS m/z (M+H⁺) 368.20.

A mixture of1-(7-benzyloxy-naphthalen-1-yl)-2-pyridin-4-yl-ethane-1,2-dione (576 mg,1.57 mmol), benzaldehyde (0.2 mL, 0.21 g, 1.98 mmol) and ammoniumacetate (1.21 g, 15.7 mmol) in acetic acid (15 mL) is stirred at 120° C.over night. The reaction mixture is then cooled to room temperature,poured into ice cold ammonium hydroxide solution and extracted withethyl acetate (3×100 mL). The organic extracts are combined, dried oversodium sulfate, filtered and concentrated in vacuum. Silica gelchromatography (CH₂Cl₂/EtOAc=2/1) of the crude product afforded4-[5-(7-benzyloxy-naphthalen-1-yl)-2-phenyl-1H-imidazol-4-yl]-pyridine(700 mg, 98% yield); ¹H NMR 400 Hz (DMSO) δ 3.30 (s, 2H), 6.99 (s, 1H),7.21 (m, 5H), 7.24 (m, 3H), 7.43 (t, J=7.31 Hz, 1H), 7.52 (t, J=7.37 Hz,3H), 7.66 (m, 1H), 8.00 (m, 2H), 8.13 (d, J=7.30 Hz, 2H), 8.32 (d,J=4.43 Hz, 2H); MS m/z (M+H⁺) 454.20.

To a solution of4-[5-(7-benzyloxy-naphthalen-1-yl)-2-phenyl-1H-imidazol-4-yl]-pyridine(810 mg, 1.79 mmol) in methanol (50 mL) and tetrahydrofuran (10 mL) isadded 10% Pd/C (100 mg) and the mixture is stirred for 3 hours underhydrogen atmosphere (1 atm). The catalyst is filtered and washed withethyl acetate. The filtrate is concentrated and silica gelchromatography (CH₂Cl₂/EtOAc=1/1) afforded8-(2-phenyl-5-pyridin-4-yl-3H-imidazol-4-yl)-naphthalen-2-ol (520 mg,80% yield) as a syrup; ¹H NMR 400 Hz (CD₃OD) δ 6.89 (d, J=2.32 Hz, 1H),7.17 (dd, J=2.38, 8.89 Hz, 1H), 7.48 (dd, J=7.15, 8.21 Hz, 1H), 7.55 (m,3H), 7.66 (dd, J=1.09, 7.04 Hz, 1H), 7.91 (d, J=8.93 Hz, 1H), 7.95 (d,J=7.03 Hz, 3H), 8.04 (d, J=8.24 Hz, 1H), 8.10 (m, 2H), 8.47 (d, J=7.02Hz, 2H); MS m/z (M+H⁺) 364.20.

A mixture of8-(2-phenyl-5-pyridin-4-yl-3H-imidazol-4-yl)-naphthalen-2-ol (31 mg,0.085 mmol), phenyl isocyanate (11 mg, 0.094 mmol) and triethylamine(17.8 μL, 12.9 mg, 0.128 mmol) in N,N-dimethylformamide (1.5 mL) isstirred for 2 hours at room temperature. Preparative LCMS affordedphenyl-carbamic acid8-(2-phenyl-5-pyridin-4-yl-3H-imidazol-4-yl)-naphthalen-2-yl ester (17mg, 41% yield); ¹H NMR 400 Hz (CD₃OD) δ 7.01 (m, 2H), 7.25 (m, 4H), 7.39(m, 5H), 7.45 (dd, J=2.28, 8.86 Hz, 1H), 7.53 (m, 3H), 7.72 (dd, J=7.14,8.23 Hz, 1H), 7.83 (d, J=6.02 Hz, 1H), 7.90 (d, J=6.99 Hz, 2H), 8.10 (m,3H), 8.19 (d, J=8.29 Hz, 1H), 8.43 (d, J=7.00 Hz, 2H); MS m/z (M+H⁺)483.20.

By repeating the procedures described in the above example, usingappropriate starting materials, the following compounds of Formula I, asidentified in Table 1, are obtained.

TABLE 1 Physical Data ¹H NMR 400 MHz Compound (DMSO-d₆) Number Structureand/or MS (m/z) 2

¹H NMR 400 Hz (DMSO) δ 7.31 (d, J = 4.40 Hz, 2 H), 7.44 (dd, J = 7.27,7.31 Hz, 1 H), 7.53 (m, 3 H), 7.61 (m, 2 H), 7.72 (m, 2 H), 8.14 (m, 4H), 8.32 (br, 2 H). MS m/z (M + H⁺) 348.10 3

¹H NMR 400 Hz (CD₃OD) δ 7.50 (m, 1 H), 7.60 (dd, J = 7.17, 7.83 Hz, 1H), 7.73 (m, 3 H), 7.94 (d, J = 6.83 Hz, 2 H), 8.06 (d, J = 7.91 Hz, 2H), 8.18 (d, J = 8.21 Hz, 1 H), 8.45 (d, J = 6.79 Hz, 2 H), 8.72 (s, 2H). MS m/z (M + H⁺) 484.10 4

¹H NMR 400 Hz (CD₃OD) δ 3.10 (s, 6 H), 6.95 (m, 2 H), 7.52 (m, 1 H),7.60 (m, 1 H), 7.72 (m, 5 H), 7.97 (d, J = 8.94 Hz, 2 H), 8.06 (d, J =8.14 Hz, 1 H), 8.18 (d, J = 8.20 Hz, 1 H), 8.49 (d, J = 6.28 Hz, 2 H).MS m/z (M + H⁺) 391.20 5

¹H NMR 400 Hz (CD₃OD) δ 3.94 (s, 3 H), 3.97 (s, 6 H), 6.46 (s, 2 H),7.63 (m, 7 H), 8.07 (d, J = 7.99 Hz, 1 H), 8.18 (d, J = 8.19 Hz, 1 H),8.52 (m, 2 H). MS m/z (M + H⁺) 438.20 6

¹H NMR 400 Hz (CD₃OD) δ 7.28 (m, 1 H), 7.38 (m, 1 H), 7.57 (m, 4 H),7.72 (m, 2 H), 7.90 (d, J = 6.99 Hz, 2 H), 8.05 (d, J = 8.13 Hz, 1 H),8.17 (m, 2 H), 8.43 (d, J = 7.02 Hz, 2 H). MS m/z (M + H⁺) 366.10 7

¹H NMR 400 Hz (CDCl₃) δ 1.43 (t, J = 6.96 Hz, 3 H), 4.27 (q, J = 6.96Hz, 2 H), 7.07 (d, J = 8.12 Hz, 1 H), 7.22 (m, 1 H), 7.44 (m, 2 H), 7.67(m, 4 H), 7.90 (d, J = 6.77 Hz, 2 H), 8.05 (d, J = 8.20 Hz, 1 H), 8.13(m, 1 H), 8.43 (d, J = 6.79 Hz, 2 H), 8.53 (dd, J = 1.74, 7.82 Hz, 1 H).MS m/z (M + H⁺) 392.20 8

¹H NMR 400 Hz (DMSO) δ 7.49 (m, 1 H), 7.67 (m, 6 H), 7.82 (t, J = 8.05Hz, 1 H), 8.09 (d, J = 7.96 Hz, 1 H), 8.18 (d, J = 7.98 Hz, 1 H), 8.27(m, 1 H), 8.52 (m, 3 H), 8.96 (t, J = 1.87 Hz, 1 H). MS m/z (M + H⁺)393.10 9

MS m/z (M + H⁺) 272.10 10

¹H NMR 400 Hz (CD₃OD) δ 7.15 (m, 2 H), 7.49 (m, 6 H), 7.68 (m, 3 H),7.86 (d, J = 5.73 Hz, 2 H), 7.91 (s, 1 H), 8.08 (d, J = 6.75 Hz, 2 H),8.22 (d, J = 5.28 Hz, 2 H). MS m/z (M + H⁺) 483.20. 11

¹H NMR 400 Hz (DMSO) δ 7.15 (m, 2 H), 7.45 (m, 1 H), 7.52 (m, 5 H), 7.81(m, 2 H), 8.01 (s, 1 H), 8.11 (d, J = 7.32 Hz, 2 H), 8.45 (br, 2 H). MSm/z (M + H⁺) 364.20 12

¹H NMR 400 Hz (CD₃OD) δ 3.89 (s, 3 H), 7.51 (m, 4 H), 7.68 (m, 3 H),7.84 (d, J = 2.0 Hz, 1 H), 8.01 (m, 3 H), 8.11 (m, 5 H), 8.23 (s, 1 H),8.56 (d, J = 6.89 Hz, 2 H). MS m/z (M + H⁺) 541.20 13

¹H NMR 400 Hz (CD₃OD) δ 5.92 (s, 2 H), 6.77 (d, J = 8.36 Hz, 1 H), 6.90(dd, J = 2.09, 8.36 Hz, 1 H), 7.17 (s, 1 H), 7.46 (dd, J = 2.29, 8.86Hz, 1 H), 7.53 (m, 3 H), 7.66 (dd, J = 1.68, 8.47 Hz, 1 H), 7.78 (d, J =2.11 Hz, 1 H), 8.09 (m, 6 H), 8.19 (s, 1 H), 8.54 (d, J = 6.97 Hz, 2 H).MS m/z (M + H⁺) 527.20 14

¹H NMR 400 Hz (CD₃OD) δ 3.92 (s, 6 H), 7.52 (m, 4 H), 7.70 (dd, J =1.64, 8.32 Hz, 1 H), 7.85 (d, J = 2.03 Hz, 1 H), 8.04 (d, J = 9.06 Hz, 1H), 8.10 (m, 5 H), 8.23 (br, 1 H), 8.34 (m, 1 H), 8.45 (d, J = 1.43 Hz,2 H), 8.55 (d, J = 7.85 Hz, 2 H). MS m/z (M + H⁺) 599.20. 15

¹H NMR 400 Hz (CD₃OD) δ 3.31 (s, 6 H), 7.53 (m, 5 H), 7.64 (d, J = 9.06Hz, 2 H), 7.70 (dd, J = 1.65, 8.47 Hz, 1 H), 7.79 (d, J = 9.09 Hz, 2 H),7.84 (d, J = 1.97 Hz, 1 H), 8.11 (m, 7 H), 8.24 (s, 1 H), 8.59 (s, br, 2H). MS m/z (M + H⁺) 526.30 16

¹H NMR 400 Hz (CD₃OD) δ 5.89 (s, 2 H), 6.72 (m, 2 H), 6.70 (s, 1 H),7.43 (m, 2 H), 7.55 (m, 3 H), 7.71 (t, J = 8.19 Hz, 1 H), 7.82 (d, J =6.17 Hz, 1 H), 7.89 (d, J = 6.97 Hz, 2 H), 8.09 (m, 3 H), 8.18 (d, J =8.23 Hz, 1 H), 8.42 (d, J = 6.96 Hz, 2 H). MS m/z (M + H⁺) 527.20 17

¹H NMR 400 Hz (CD₃OD) δ 3.92 (s, 6 H), 7.43 (dd, J = 2.23, 8.84 Hz, 1H), 7.50 (m, 4 H), 7.74 (dd, J = 7.20, 8.20 Hz, 1 H), 7.90 (m, 3 H),8.10 (m, 3 H), 8.19 (d, J = 8.29 Hz, 1 H), 8.25(m, 3 H), 8.49 (d, J =6.94 Hz, 2 H). MS m/z (M + H⁺) 599.10 18

¹H NMR 400 Hz (CD₃OD) δ 3.24 (s, 6 H), 7.47 (m, 2 H), 7.54 (m, 5 H),7.61 (m, 2 H), 7.73 (dd, J = 7.38, 8.20 Hz, 1 H), 7.83 (m, 1 H), 7.91(d, J = 6.98 Hz, 2 H), 8.11 (m, 3 H), 8.20 (d, J = 8.28 Hz, 1 H), 8.46(d, J = 6.96 Hz, 2 H). MS m/z (M + H⁺) 526.30

Assays

Compounds of the present invention are assayed to measure their capacityto selectively inhibit cell proliferation of 32D cells expressingBCR-Abl (32D-p210) compared with parental 32D cells. Compoundsselectively inhibiting the proliferation of these BCR-Abl transformedcells are tested for anti-proliferative activity on Ba/F3 cellsexpressing either wild type or the mutant forms of Bcr-abl. In addition,compounds are assayed to measure their capacity to inhibit b-Raf.

Inhibition of Cellular BCR-Abl Dependent Proliferation (High ThroughputMethod)

The murine cell line used is the 32D hemopoietic progenitor cell linetransformed with BCR-Abl cDNA (32D-p210). These cells are maintained inRPMI/10% fetal calf serum (RPMI/FCS) supplemented with penicillin 50μg/mL, streptomycin 50 μg/mL and L-glutamine 200 mM. Untransformed 32Dcells are similarly maintained with the addition of 15% of WEHIconditioned medium as a source of IL3.

50 μl of a 32D or 32D-p210 cells suspension are plated in Greiner 384well microplates (black) at a density of 5000 cells per well. 50 nl oftest compound (1 mM in DMSO stock solution) is added to each well(STI571 is included as a positive control). The cells are incubated for72 hours at 37° C., 5% CO₂. 10 μl of a 60% Alamar Blue solution (Tekdiagnostics) is added to each well and the cells are incubated for anadditional 24 hours. The fluorescence intensity (Excitation at 530 nm,Emission at 580 nm) is quantified using the Acquest™ system (MolecularDevices).

Inhibition of Cellular BCR-Abl Dependent Proliferation

32D-p210 cells are plated into 96 well TC plates at a density of 15,000cells per well. 50 μL of two fold serial dilutions of the test compound(C_(max) is 40 μM) are added to each well (STI571 is included as apositive control). After incubating the cells for 48 hours at 37° C., 5%CO₂, 15 μL of MTT (Promega) is added to each well and the cells areincubated for an additional 5 hours. The optical density at 570 nm isquantified spectrophotometrically and IC₅₀ values, the concentration ofcompound required for 50% inhibition, determined from a dose responsecurve.

Effect on Cell Cycle Distribution

32D and 32D-p210 cells are plated into 6 well TC plates at 2.5×10⁶ cellsper well in 5 ml of medium and test compound at 1 or 10 μM is added(STI571 is included as a control). The cells are then incubated for 24or 48 hours at 37° C., 5% CO₂. 2 ml of cell suspension is washed withPBS, fixed in 70% EtOH for 1 hour and treated with PBS/EDTA/RNase A for30 minutes. Propidium iodide (Cf=10 μg/ml) is added and the fluorescenceintensity is quantified by flow cytometry on the FACScalibur™ system (BDBiosciences). Test compounds of the present invention demonstrate anapoptotic effect on the 32D-p210 cells but do not induce apoptosis inthe 32D parental cells.

Effect on Cellular BCR-Abl Autophosphorylation

BCR-Abl autophosphorylation is quantified with capture Elisa using ac-abl specific capture antibody and an antiphosphotyrosine antibody.32D-p210 cells are plated in 96 well TC plates at 2×10⁵ cells per wellin 50 μL of medium. 50 μL of two fold serial dilutions of test compounds(C_(max) is 10 μM) are added to each well (STI571 is included as apositive control). The cells are incubated for 90 minutes at 37° C., 5%CO₂. The cells are then treated for 1 hour on ice with 150 μL of lysisbuffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM EGTA and 1%NP-40) containing protease and phosphatase inhibitors. 50 μL of celllysate is added to 96 well optiplates previously coated with anti-ablspecific antibody and blocked. The plates are incubated for 4 hours at4° C. After washing with TBS-Tween 20 buffer, 50 μL ofalkaline-phosphatase conjugated anti-phosphotyrosine antibody is addedand the plate is further incubated overnight at 4° C. After washing withTBS-Tween 20 buffer, 90 μL of a luminescent substrate are added and theluminescence is quantified using the Acquest™ system (MolecularDevices). Test compounds of the invention that inhibit the proliferationof the BCR-Abl expressing cells, inhibit the cellular BCR-Ablautophosphorylation in a dose-dependent manner.

Effect on Proliferation of Cells Expressing Mutant Forms of Bcr-Abl

Compounds of the invention are tested for their antiproliferative effecton Ba/F3 cells expressing either wild type or the mutant forms ofBCR-Abl (G250E, E255V, T315I, F317L, M351T) that confers resistance ordiminished sensitivity to STI571. The antiproliferative effect of thesecompounds on the mutant-BCR-Abl expressing cells and on the nontransformed cells were tested at 10, 3.3, 1.1 and 0.37 μM as describedabove (in media lacking IL3). The IC₅₀ values of the compounds lackingtoxicity on the untransformed cells were determined from the doseresponse curves obtained as describe above.

b-Raf

Compounds of the invention are tested for their ability to inhibit theactivity of b-Raf. The assay is carried out in 384-well MaxiSorp plates(NUNC) with black walls and clear bottom. The substrate, IκBα is dilutedin DPBS (1:750) and 15 μl is added to each well. The plates areincubated at 4° C. overnight and washed 3 times with TBST (25 mM Tris,pH 8.0, 150 mM NaCl and 0.05% Tween-20) using the EMBLA plate washer.Plates are blocked by Superblock (15 μl/well) for 3 hours at roomtemperature, washed 3 times with TBST and pat-dried. Assay buffercontaining 20 μM ATP (10 μl) is added to each well followed by 100 nl or500 nl of compound. B-Raf is diluted in the assay buffer (1 μl into 25μl) and 10 μl of diluted b-Raf is added to each well (0.4 μg/well). Theplates are incubated at room temperature for 2.5 hours. The kinasereaction is stopped by washing the plates 6 times with TBST. Phosph-IκBα(Ser32/36) antibody is diluted in Superblock (1:10,000) and 15 μl isadded to each well. The plates are incubated at 4° C. overnight andwashed 6 times with TBST. AP-conjugated goat-anti-mouse IgG is dilutedin Superblock (1:1,500) and 15 μl is added to each well. Plates areincubated at room temperature for 1 hour and washed 6 times with TBST.15 μl of Attophos AP substrate is added to each well and plates areincubated at room temperature for 15 minutes. Plates are read on Acquestor Analyst GT using a Fluorescence Intensity Nanxin BBT anion (505dichroic mirror).

Upstate KinaseProfiler™-Radio-Enzymatic Filter Binding Assay

Compounds of the invention are assessed for their ability to inhibitindividual members of a panel of kinases (a partial, non-limiting listof kinases includes: Abl, BCR-Abl, EGF-R, c-erbB2 kinase (HER-2),PDGF-R, lck, SAPK2α, p38, TGFβ, KDR, c-Kit, b-RAF, c-RAF, FLT1 andFLT4). The compounds are tested in duplicates at a final concentrationof 10 μM following this generic protocol. Note that the kinase buffercomposition and the substrates vary for the different kinases includedin the “Upstate KinaseProfiler™” panel. The compounds are tested induplicates at a final concentration of 10 μM following this genericprotocol. Note that the kinase buffer composition and the substratesvary for the different kinases included in the “Upstate KinaseProfiler™”panel. Kinase buffer (2.5 μL, 10×-containing MnCl₂ when required),active kinase (0.001-0.01 Units; 2.5 μL), specific or Poly (Glu4-Tyr)peptide (5-500 μM or 0.01 mg/ml) in kinase buffer and kinase buffer (50μM; 5 μL) are mixed in an eppendorf on ice. A Mg/ATP mix (10 μL; 67.5(or 33.75) mM MgCl₂, 450 (or 225) μM ATP and 1 μCi/μl [γ-³²P]-ATP (3000Ci/mmol)) is added and the reaction is incubated at about 30° C. forabout 10 minutes. The reaction mixture is spotted (20 μL) onto a 2 cm×2cm P81 (phosphocellulose, for positively charged peptide substrates) orWhatman No. 1 (for Poly (Glu4-Tyr) peptide substrate) paper square. Theassay squares are washed 4 times, for 5 minutes each, with 0.75%phosphoric acid and washed once with acetone for 5 minutes. The assaysquares are transferred to a scintillation vial, 5 ml scintillationcocktail are added and ³²P incorporation (cpm) to the peptide substrateis quantified with a Beckman scintillation counter. Percentageinhibition is calculated for each reaction.

Compounds of Formula I, in free form or in pharmaceutically acceptablesalt form, exhibit valuable pharmacological properties, for example, asindicated by the in vitro tests described in this application. Forexample, compounds of Formula I preferably show an IC₅₀ in the range of1×10⁻¹⁰ to 1×10⁻⁵ M, preferably less than 500 nM for wild type BCR-Abland b-Raf. For example,6-(2-phenyl-5-pyridin-4-yl-1H-imidazol-4-yl)-naphthalen-2-ol (compound11) and (4-dimethylamino-phenyl)-carbamic acid6-(2-phenyl-5-pyridin-4-yl-1H-imidazol-4-yl)-naphthalen-2-yl ester(compound 15) have an IC₅₀ of 300 nM and 879 nM against b-Raf,respectively.

Compounds of Formula I, at a concentration of 10 μM, preferably show apercentage inhibition of greater than 50%, preferably greater than about70%, against Abl, BCR-Abl, PDGF-R, lck, SAPK20α, p38, TGFβ, KDR, c-Kit,b-RAF, c-RAF, FLT1 and/or FLT4 kinases. For example,6-(2-Phenyl-5-pyridin-4-yl-1H-imidazol-4-yl)-naphthalen-2-ol (compound11) at a concentration of 10 μM, inhibits the following kinases by thepercentage shown in brackets (for example, 100% means completeinhibition, 0% means no inhibition): Abl (98%); c-RAF (98%), Lck (61%);and SAPK2α (74%).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference for allpurposes.

1. A compound of Formula I:

in which: R₁ is selected from hydrogen, —XR₃, —XC(O)R₃, —XSR₃, —XS(O)R₃,—XS(O)₂R₃, —XOR₄, and —XOC(O)NR₃R₄; wherein X is a bond or C₁₋₄alkylene;R₂ is selected from —XR₃, —XC(O)R₃, —XSR₃, and —XOC(O)NR₃R₄; wherein Xis a bond or C₁₋₄alkylene; R₄ is selected from hydrogen and C₁₋₆alkyl;R₃ is selected from C₅₋₁₀heteroaryl, and C₃₋₈heterocycloalkyl; whereinany heteroaryl, or heterocycloalkyl of R₃ is optionally substituted by 1to 3 radicals selected from hydroxy, halo, nitro, C₁₋₆alkyl, C₁₋₆alkoxy,halo-substituted-C₁₋₆alkyl, halo-substituted-C₁₋₆alkoxy, —XC(O)OR₄ and—NR₄R₅; wherein X is a bond or C₁₋₄alkylene and R₄ and R₅ areindependently selected from hydrogen and C₁₋₆alkyl; and pharmaceuticallyacceptable salts thereof.
 2. The compound of claim 1, wherein: R₁ isselected from hydrogen, —XR₃, and —XOC(O)NR₃R₄; wherein X is a bond orC₁₋₄alkylene; R₂ is selected from —XR₃ and —XOC(O)NR₃R₄; wherein X is abond or C₁₋₄alkylene; R₄ is selected from hydrogen and C₁₋₆alkyl; R₃ isC₅₋₁₀heteroaryl or C₃₋₈heterocycloalkyl optionally substituted by 1 to 3radicals selected from halo, nitro, C₁₋₆alkyl, C₁₋₆alkoxy,halo-substituted-C₁₋₆alkyl, —XC(O)OR₄ and —NR₄R₅; wherein X is a bond orC₁₋₄alkylene and R₄ and R₅ are independently selected from hydrogen andC₁₋₆alkyl.
 3. The compound of claim 2, wherein R₁ is selected fromhydrogen and phenyl; wherein said phenyl is optionally substituted by 1to 3 radicals selected from trifluoromethyl, dimethylamino, methoxy,halo, ethoxy and nitro.
 4. The compound of claim 2, wherein in which R₂is —OC(O)NHR₃ and R₃ is benzo[1,3]dioxol-5-yl optionally substituted by1 to 3 radicals selected from —C(O)OCH₃ and dimethylamino.
 5. Thecompound of claim 1 selected from benzo[1,3]dioxol-5-yl-carbamic acid6-(2-phenyl-5-pyridin-4-yl-1H-imidazol-4-yl)-naphthalen-2-yl ester andbenzo[1,3]dioxol-5-yl-carbamic acid8-(2-phenyl-5-pyridin-4-yl-1H-imidazol-4-yl)-naphthalen-2-yl ester.
 6. Apharmaceutical composition comprising a therapeutically effective amountof a compound of claim 1 in combination with a pharmaceuticallyacceptable excipient.